Our long-term objective is to understand the regulation of different Th subsets which can and will develop in response to Ag and to identify the factors which determine the size and type of each population. We will use a TcR transgenic mouse model in which we can generate populations of Ag- specific CD4 T cells that are naive, Th2 or Th2 effectors or resting Th1 or Th2 memory cells using procedures developed in the previous grant periods. In vitro, we will generate 4 day effectors that have ThO (IL-2, IL-4, IFNgamma), Th1 (IL-2, IFNgamma) or Th2 (IL-4, IL-10) patterns of cytokine secretion and we will generate effector subsets in vivo, after transfer of naive CD4 to adoptive hosts, followed by immunization of the host. We will transfer polarized effectors to adoptive hosts to obtain memory cells with related cytokine polarization. We will use vital dyes and FACS analysis to track transferred cells in adoptive hosts and to visualize their fate including an analysis of cell division and phenotypic change. We will assess the functions of in vivo generated subsets both in situ and after re-isolation and in vitro restimulation. We will investigate the cytokines, co-stimulatory pathways and Ag-presenting cell populations and Ag doses/numbers of encounters which are required to generate each subset and to elicit cytokine production from and further expansion of each subset. We will particularly focus on apoptosis as a mechanism regulating the size and persistence of populations of different subsets. We will continue to seek the mechanisms which protect Th2 effectors from cell death, and which enable IL-2 and TGFbeta to protest cells from undergoing apoptosis. We will include in vivo studies to evaluate the impact of apoptosis as an in situ regulator of the dynamics of lymphocytes during immune response. Overall these Aims should provide important indications of the host factors that are likely to influence the initial and long-term generation of distinct Th subsets. We believe that the nature and amount of Th help which develops during a response to a particular Ag will play the major role in determining both the magnitude of the ensuing immune response and the kind of the immune response that occurs. Understanding how the generation of Th subsets is itself regulated, should give us clues about potential therapeutic intervention to influence development of particular subsets when appropriate immune responses fail to develop or when ineffective or even counterproductive immune responses occur. Furthermore, as we gain a greater understanding of the in vivo persistence and function of Th subsets that have been generated in vitro and transferred to adoptive hosts, we may be able to devise well focused protocols for stimulating autologous T cells in vitro and then reintroducing them to achieve desired immunity.